Seed coat specific DNA regulatory region and peroxidase

ABSTRACT

A novel seed coat specific peroxidase genomic sequence is characterized and presented. The seed coat peroxidase is translated as a 352 amino acid precursor protein of 38 kDa comprising a 26 amino acid signal sequence which when cleaved results in a 35 kDa protein. Probes derived from the cDNA, or genomic DNA can be used to detect polymorphisms that distinguish EpEp and epep genotypes. The regulatory region of the seed coat specific gene may be used to control expression of genes of interest such as genes encoding herbicide resistance, biological control of insects or pathogens, viral coat proteins to protect against viral infections, proteins of commercial interest, or proteins that alter the nutritive value, taste, or processing of seeds.

The present invention relates to a novel DNA molecule comprising a plant seed coat specific DNA regulatory region and a novel structural gene encoding a peroxidase. The seed-coat specific DNA regulatory region may also be used to control the expression of other genes of interest within the seed coat.

BACKGROUND OF THE INVENTION

Full citations for references appear at the end of the Examples section.

Peroxidases are enzymes catalyzing oxidative reactions that use H₂O₂ as an electron acceptor. These enzymes are widespread and occur ubiquitously in plants as isozymes that may be distinguished by their isoelectric points. Plant peroxidases contribute to the structural integrity of cell walls by functioning in lignin biosynthesis and suberization, and by forming covalent cross-linkages between extension, cellulose, pectin and other cell wall constituents (Campa, 1991). Peroxidases are also associated with plant defence responses and resistance to pathogens (Bowles, 1990; Moerschbacher 1992). Soybeans contain 3 anionic isozymes of peroxidase with a minimum M_(r) of 37 kDa (Sessa and Anderson, 1981). Recently one peroxidase isozyme, localised within the seed coat of soybean, has been characterized with a M_(r) of 37 kDa (Gillikin and Graham, 1991).

In an analysis of soybean seeds, Buttery and Buzzell (1968) showed that the amount of peroxidase activity present in seed coats may vary substantially among different cuitivars. The presence of a single dominant gene Ep causes a high seed coat peroxidase phenotype (Buzzell and Buttery, 1969). Homozygous recessive epep plants are ˜100-fold lower in seed coat peroxidase activity. This results from a reduction in the amount of peroxidase enzyme present, primarily in the hourglass cells of the subepidermis (Gijzen et al., 1993). In plants carrying the Ep gene, peroxidase is heavily concentrated in the hourglass cells (osteosclereids). These cells form a highly differentiated cell layer with thick, elongated secondary walls and large intercellular spaces (Baker et al., 1987). Hourglass cells develop between the epidermal macrosclereids and the underlying articulated parenchyma, and are a prominent feature of seed coat anatomy at full maturity. The cytoplasm exudes from the hourglass cells upon imbibition with water and a distinct peroxidase isozyme constitutes five to 10% of the total soluble protein in EpEp seed coats. It is not known why the hourglass cells accumulate large amounts of peroxidase, but the sheer abundance and relative purity of the enzyme in soybean seed coats is significant because peroxidases are versatile enzymes with many commercial and industrial applications. Studies of soybean seed coat peroxidase have shown this enzyme to have useful catalytic properties and a high degree of thermal stability even at extremes of pH (McEldoon et al., 1995). These properties result in the preferred use of soybean peroxidase, over that of horseradish peroxidase, in diagnostic assays as an enzyme label for antigens, antibodies, oligonucleotide probes, and within staining techniques. Johnson et al report on the use of soybean peroxidase for the deinking of printed waste paper (U.S. Pat. No. 5,270,770; Dec. 6, 1994) and for the biocatalytic oxidation of primary alcohols (U.S. Pat. No. 5,391,488; Feb. 13, 1996). Soybean peroxidase has also been used as a replacement for chlorine in the pulp and paper industry, or as formaldehyde replacement (Freiberg, 1995).

An anionic soybean peroxidase from seed coats has been purified (Gillikin and Graham, 1991). This protein has a pI of 4.1 and Mr of 37 kDa. A method for the bulk extraction of peroxidase from seed hulls of soybean using a freeze thaw technique has also been reported (U.S. Pat. No. 5,491,085, Feb. 13, 1996, Pokara and Johnson).

Lagrimini et al (1987) disclose the cloning of a ubiquitous anionic peroxidase in tobacco encoding a protein of Mr of 36 kDa. This peroxidase has also been over expressed in transgenic tobacco plants (Lagrimini et al 1990) and Maliyakal discloses the expression of this gene in cotton (WO 95/08914).

Huangpu et al (1995) reported the partial cloning of a soybean anionic seed coat peroxidase. The 1031 bp sequence contained an open reading frame of 849 bp encoding a 283 amino acid protein with a Mr of 30,577. The M_(r) of this peroxidase is 7 kDa less than what one would expect for a soybean seed coat peroxidase as reported by Gillikin and Graham (1991) and possibly represents another peroxidase isozyme within the seed coat.

The upstream promoter sequences for two poplar peroxidases have been described by Osakabe et al (1995). A number of characteristic regulatory sites were identified from comparison of these sequences to existing promoter elements. Additionally, a cryptic promoter with apparent specificity for seed coat tissues was isolated from tobacco by a promoter trapping strategy (Fobert et al. 1994). The upstream regulatory sequences associated with the Ep gene in soybean are distinct from these and other previously characterized promoters. The soybean Ep promoter drives high-level expression in a cell and tissue specific manner. The peroxidase protein encoded by the Ep gene accumulates in the seed coat tissues, especially in the hour glass cells of the subepidermis. Minimal expression of the gene is detected in root tissues.

One problem arising from the desired use of soybean seed coat peroxidase is that there is variability between soybean varieties regarding peroxidase production (Buttery and Buzzell, 1986; Freiberg, 1995). Due to the commercial interest in the use of soybean seed coat peroxidase new methods of producing this enzyme are required. Therefore, the gene responsible for the expression of the 37 kDa isozyme in soybean seed coat was isolated and characterized.

Furthermore, novel regulatory regions obtained from the genomic DNA of soybean seed coat peroxidase have been isolated and characterized and are useful in directing the expression of genes of interest in seed coat tissues.

SUMMARY OF THE INVENTION

The present invention relates to a DNA molecule that encodes a soybean seed coat peroxidase and associated DNA regulatory regions.

This invention also embraces isolated DNA molecules comprising the nucleotide sequence of either SEQ ID NO:1 (the cDNA encoding soybean seed coat peroxidase) SEQ ID No:2 (the genomic sequence).

This invention also provides for a chimeric DNA molecule comprising a seed coat-specific regulatory region having nucleotides 1-1532 of SEQ ID NO:2 and a gene of interest under control of this DNA regulatory region. Also included within this invention are chimeric DNA molecules comprising genomic DNA sequences exemplified by nucleotides 412-1041, 1234-2263 or 2430-2691 of SEQ ID NO:2. Furthermore, this invention is directed to isolated DNA molecules comprising at least

-   -   1) 24 contiguous nucleotides selected from nucleotides 1-1532 of         SEQ ID NO:2;     -   2) 32 contiguous nucleotides selected from nucleotides 412-1041         of SEQ ID NO:2;     -   3) 23 contiguous nucleotides selected from nucleotides 1234-2263         of SEQ ID NO:2; or     -   4) 22 contiguous nucleotides selected from nucleotides 2430-2691         of SEQ ID NO:2.

The present invention also provides for vectors which comprise DNA molecules encoding soybean seed coat peroxidase. Such a construct may include the DNA regulatory region from SEQ ID NO:2, including nucleotides 1-1532, or at least 24 contiguous nucleotides selected from nucleotides 1-1532 of SEQ ID NO:2 in conjunction with the seed coat peroxidase gene, or the seed coat peroxidase gene under the control of any suitable constitutive or inducible promoter of interest.

This invention is also directed towards vectors which comprise a gene of interest placed under the control of a DNA regulatory element derived from the genomic sequence encoding soybean seed coat peroxidase. Such a regulatory element includes nucleotides 1-1532 of SEQ ID NO:2, or at least 24 contiguous nucleotides selected from nucleotides 1-1532 of SEQ ID NO:2. Elements comprising nucleotides 412-1041, 1234-2263 or 2430-2691 of SEQ ID NO:2, or 32 contiguous nucleotides selected from nucleotides 412-1041 of SEQ ID NO:2, 23 contiguous nucleotides selected from nucleotides 1234-2263 of SEQ ID NO:2, or 22 contiguous nucleotides selected from nucleotides 2430-2691 of SEQ ID NO:2 may also be used.

This invention also embraces prokaryotic and eukaryotic cells comprising the vectors identified above. Such cells may include bacterial, insect, mammalian, and plant cell cultures.

This invention also provides for transgenic plants comprising the seed coat peroxidase gene under control of constitutive or inducible promoters. Furthermore, this invention also relates to transgenic plants comprising the DNA regulatory regions of nucleotides 1-1532 of SEQ ID NO:2 controlling a gene of interest, or comprising genes of interest in functional association with genomic DNA sequences exemplified by nucleotides 412-1041, 1234-2263 or 2430-2691 of SEQ ID NO:2. Also embraced by this invention are transgenic plants having regulatory regions comprising at least 24 contiguous nucleotides selected from nucleotides 1-1532 of SEQ ID NO:2, 32 contiguous nucleotides selected from nucleotides 412-1041 of SEQ ID NO:2, 23 contiguous nucleotides selected from nucleotides 1234-2263 of SEQ ID NO:2, or 22 contiguous nucleotides selected from nucleotides 2430-2691 of SEQ ID NO:2.

This invention is also directed to a method for the production of soybean seed coat peroxidase in a host cell comprising:

-   -   i) transforming the host cell with a vector comprising an         oligonucleotide sequence that encodes soybean seed coat         peroxidase; and     -   ii) culturing the host cell under conditions to allow expression         of the soybean seed coat peroxidase.

This invention also provides for a process for producing a heterologous gene of interest within seed coats of a transformed plant, comprising propagating a plant transformed with a vector comprising a gene of interest under the control of nucleotides 1-1532 of SEQ ID NO:2. Furthermore, this invention embraces a process for producing a heterologous gene of interest within seed coats of a transformed plant, comprising propagating a plant transformed with a vector comprising a gene of interest under the control of a regulatory region comprising at least 24 nucleotides selected from nucleotides 1-1532 of SEQ ID NO:2.

Although the present invention is exemplified by a soybean seed coat peroxidase and adjacent DNA regulatory regions, in practice any gene of interest can be placed downstream from the DNA regulatory region for seed coat specific expression.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

FIG. 1 is the cDNA and deduced amino acid sequence of soybean seed coat peroxidase. Nucleotides are numbered by assigning +1 to the first base of the ATG start codon; amino acids are numbered by assigning +1 to the N-terminal Gln residue after cleavage of the putative signal sequence. The N-terminal signal sequence, the region of the active site, and the heme-binding domain are underlined. The numerals I, II and III placed directly above single nucleotide gaps in the sequence indicate the three intron splice positions. The target site and direction of five different PCR primers are shown with dotted lines above the nucleotide sequence. An asterisk (*) marks the translation stop codon.

FIG. 2 is the genomic DNA sequence of the Soybean seed coat peroxidase.

FIG. 3 is a comparison of soybean seed coat peroxidase with other closely related plant peroxidases. The GenBank accession numbers are provided next to the name of the plant from which the peroxidase was isolated. The accession number for the soybean sequence is L78163. (A) A comparison of the nucleic acid sequences; (B) A comparison of the amino acid sequences.

FIG. 4 is a restriction fragment length polymorphisms between EpEp and epep genotypes using the seed coat peroxidase cDNA as probe. Genomic DNA of soybean lines OX312 (epep) and OX347 (EpEp) was digested with restriction enzyme, separated by electrophoresis in a 0.5% agarose gel, transferred to nylon, and hybridized with ³²P-labelled cDNA encoding the seed coat peroxidase. The size of the hybridizing fragments was estimated by comparison to standards and is indicated on the right.

FIG. 5 exhibits the structure of the Ep Locus. A 17 kb fragment including the Ep locus is illustrated schematically. A 3.3 kb portion of the gene is enlarged and exons and introns are represented by shaded and open boxes, respectively. The final enlargement of the 5′ region shows the location and DNA sequence around the 87 bp deletion occurring in the ep allele of soybean line OX312. Nucleotides are numbered by assigning +1 to the first base of the ATG start codon.

FIG. 6 displays PCR analysis of EpEp and epep genotypes using primers derived from the seed coat peroxidase cDNA. Genomic DNA from soybean lines OX312 (epep) and OX347 (EpEp) was used as template for PCR analysis with four different primer sets. Amplification products were separated by electrophoresis through a 0.8% agarose gel and visualized under UV light after staining with ethidium bromide. Genotype and primer combinations are indicated at the top of the figure. The size in base pairs of the amplified DNA fragments are indicated on the right.

FIG. 7 exhibits PCR analysis of an F2 population from a cross of EpEp and epep genotypes. Genomic DNA was used as template for PCR analysis of the parents (P) and 30 F₂ individuals. The cross was derived from the soybean lines OX312 (epep) and OX347 (EpEp). Plants were self pollinated and seeds were collected and scored for seed coat peroxidase activity. The symbols (−) and (+) indicate low and high seed coat peroxidase activity, respectively. Primers prx9+ and prx10− were used in the amplification reactions. Products were separated by electrophoresis through a 0.8% agarose gel and visualized under UV light after staining with ethidium bromide. The migration of molecular markers and their corresponding size in kb is also shown (lanes M).

FIG. 8 displays PCR analysis of six different soybean cultivars with primers derived from the seed coat peroxidase cDNA sequence. Genomic DNA was used as template for. PCR analysis of three EpEp cultivars and three epep cultivars. Primers used in the amplification reactions and the size of the DNA product is indicated on the left. Products were separated by electrophoresis through a 0.8% agarose gel and visualized under UV light after staining with ethidium bromide.

-   -   (A) Forward and reverse primers are downstream from deletion     -   (B) Forward primer anneals to site within deletion     -   (C) Primers span deletion

FIG. 9 shows the accumulation of peroxidase RNA in tissues of GEp and epep plants. FIG. 9(A): A comparison of peroxidase transcript abundance in cultivars Harosoy 63 (Ep) or Marathon (ep). Seed and pod tissues were sampled at a late stage of development corresponding to a whole seed fresh weight of 250 mg. Root and leaf tissue was from six week old plants. Autoradiograph exposed for 96 h. FIG. 9(B): Developmental expression of peroxidase in cultivar Harosoy 63 (Ep). Flowers were sampled immediately after opening. Seed coat tissues were sampled at four stages of development corresponding to a whole seed fresh weight of: lane 1, 50 mg; lane 2, 100 mg; lane 3, 200 mg; lane 4, 250 mg. Autoradiograph exposed for 20 h.

DESCRIPTION OF PREFERRED EMBODIMENT

The present invention is directed to a novel oligonucleotide sequence encoding a seed coat peroxidase and associated DNA regulatory regions.

According to the present invention DNA sequences that are “substantially homologous” includes sequences that are identified under conditions of high stringency. “High stringency” refers to Southern hybridization conditions employing washes at 65° C. with 0.1×SSC, 0.5% SDS.

By “DNA regulatory region” it is meant any region within a genomic sequence that has the property of controlling the expression of a DNA sequence that is operably linked with the regulatory region. Such regulatory regions may include promoter or enhancer regions, and other regulatory elements recognized by one of skill in the art. A segment of the DNA regulatory region is exemplified in this invention, however, as is understood by one of skill in the art, this region may be used as a probe to identify surrounding regions involved in the regulation of adjacent DNA, and such surrounding regions are also included within the scope of this invention.

In the context of this disclosure, the term “promoter” or “promoter region” refers to a sequence of DNA, usually upstream (5′) to the coding sequence of a structural gene, which controls the expression of the coding region by providing the recognition for RNA polymerase and/or other factors required for transcription to start at the correct site.

There are generally two types of promoters, inducible and constitutive. An “inducible promoter” is a promoter that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer the DNA sequences or genes will not be transcribed. Typically the protein factor, that binds specifically to an inducible promoter to activate transcription, is present in an inactive form which is then directly or indirectly converted to the active form by the inducer. The inducer can be a chemical agent such as a protein, metabolite, growth regulator, herbicide or phenolic compound or a physiological stress imposed directly by heat, cold, salt, or toxic elements or indirectly through the action of a pathogen or disease agent such as a virus. A plant cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating or similar methods.

By “constitutive promoter” it is meant a promoter that directs the expression of a gene throughout the various parts of a plant and continuously throughout plant development. Examples of known constitutive promoters include those associated with the CaMV 35S transcript and Agrobacterium Ti plasmid nopaline synthase gene.

The chimeric gene constructs of the present invention can further comprise a 3′ untranslated region. A 3′ untranslated region refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by effecting the addition of polyadenylic acid tracks to the 3′ end of the mRNA precursor. Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5′ AATAAA-3′ although variations are not uncommon.

Examples of suitable 3′ regions are the 3′ transcribed non-translated regions containing a polyadenylation signal of Agrobacterium tumour inducing (Ti) plasmid genes, such as the nopaline synthase (Nos gene) and plant genes such as the soybean storage protein genes and the small subunit of the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO) gene. The 3′ untranslated region from the structural gene of the present construct can therefore be used to construct chimeric genes for expression in plants.

The chimeric gene construct of the present invention can also include further enhancers, either translation or transcription enhancers, as may be required. These enhancer regions are well known to persons skilled in the art, and can include the ATG initiation codon and adjacent sequences. The initiation codon must be in phase with the reading frame of the coding sequence to ensure translation of the entire sequence. The translation control signals and initiation codons can be from a variety of origins, both natural and synthetic. Translational initiation regions may be provided from the source of the transcriptional initiation region, or from the structural gene. The sequence can also be derived from the promoter selected to express the gene, and can be specifically modified so as to increase translation of the mRNA.

To aid in identification of transformed plant cells, the constructs of this invention may be further manipulated to include plant selectable markers. Useful selectable markers include enzymes which provide for resistance to an antibiotic such as gentamycin, hygromycin, kanamycin, and the like. Similarly, enzymes providing for production of a compound identifiable by colour change such as GUS (β-glucuronidase), or luminescence, such as luciferase are useful.

Also considered part of this invention are transgenic plants containing the chimeric gene construct of the present invention. Methods of regenerating whole plants from plant cells are known in the art, and the method of obtaining transformed and regenerated plants is not critical to this invention. In general, transformed plant cells are cultured in an appropriate medium, which may contain selective agents such as antibiotics, where selectable markers are used to facilitate identification of transformed plant cells. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be used to establish repetitive generations, either from seeds or using vegetative propagation techniques.

The constructs of the present invention can be introduced into plant cells using-Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, micro-injection, electroporation, etc. For reviews of such techniques see for example Weissbach and Weissbach (1988) and Geierson and Corey (1988). The present invention further includes a suitable vector comprising the chimeric gene construct.

Buttery and Buzzell (1968) showed that the amount of peroxidase activity present in seed coats may vary substantially among different cultivars. The presence of a single dominant gene Ep causes a high seed coat peroxidase phenotype (Buzzell and Buttery, 1969). Homozygous recessive epep plants are ˜100-fold lower in seed coat peroxidase activity. This results from a reduction in the amount of peroxidase enzyme present, primarily in the hourglass cells of the subepidermis (Gijzen et al., 1993). In plants carrying the Ep gene, peroxidase is heavily concentrated in the hourglass cells (osteosclereids). These cells form a highly differentiated cell layer with thick, elongated secondary walls and large intercellular spaces (Baker et al., 1987).

Screening a seed coat cDNA library prepared from EpEp plants with a degenerate primer derived from the active site domain of plant peroxidase resulted in a high frequency of positive clones. Many of these clones encode identical cDNA molecules and indicate that the corresponding mRNA is an abundant transcript in developing seed coat tissues. The sequence of the cDNA is shown in FIG. 1.

Previous studies on soybean seed coat peroxidase indicated that this enzyme is heavily glycosylated and that carbohydrate contributes 18% of the mass of the apo-enzyme (Gray et al., 1996). The seven potential glycosylation sites identified from the amino acid sequence of the seed cost peroxidase (FIG. 1) would accommodate the five or six N-linked glycosylation sites proposed by Gray et al. (1996). The heme-binding domain encompasses residues Asp161 to Phe171 and the acid-base catalysis region from Gly33 to Cys44. The two regions are highly conserved among plant peroxidases and are centred around functional histidine residues, His169 and His40. There are eight conserved cysteine residues in the mature protein that provide for four di-sulfide bridges found in other plant peroxidases and predicted from the crystal structure of peanut peroxidase (Welinder, 1992; Schuller et al., 1996). Other conserved areas include residues Cys91 to Ala105 and Val119 to Leu127 that occur in or around helix D. The most divergent aspects of the seed coat peroxidase protein sequence are the carboxy- and amino-terminal regions. These sequences probably provide special targeting signals for the proper processing and delivery of the peptide chain. It is possible the carboxy-terminal extension of the seed coat peroxidase is removed at maturity, as has been shown for certain barley and horseradish peroxidases (Welinder, 1992).

The molecular mass of the enzyme has been determined by denaturing gel electrophoresis to be 37 kDa (Sessa and Anderson, 1981; Gillikin and Graham, 1991) or 43 kDa (Gijzen et al., 1993). Analysis by mass spectrometry indicated: a mass of 40,622 Da for the apo-enzyme and 33,250 Da after deglycosylation (Gray et al., 1996). These values are in good agreement with the mass of 35,377 Da calculated from the predicted amino acid sequence for the mature apo-protein prior to glycosylation and other modifications. Huangpu et al (1995) reported an anionic seed coat peroxidase having a Mr of 30,577 Da and characterized a partial cDNA encoding this protein. This 1031 bp cDNA contained an open reading frame of 849 bp encoding a 283 amino acid protein. There are several differences between this reported sequence and the sequence of this invention that are manifest at the amino acid level (see FIG. 3 for sequence comparison). The enzyme encoded by the gene reported by Huangpu et al is different from that of this invention as the peroxidase of this invention has a Mr of 35,377 Da.

Genomic DNA blots probed with the seed coat peroxidase cDNA produced two or three hybridizing fragments of varying intensity with most restriction enzyme digestions, despite that several peroxidase isozymes are present in soybean. The results indicate that this seed coat peroxidase is present as a single gene that does not share sufficient homology with most other peroxidase genes to anneal under conditions of high stringency.

The genomic DNA sequence comprises four exons spanning bp 1533-1752 (exon I), 2383-2574 (exon 2), 3605-3769 (exon 3) and 40334516 (exon 4) and three introns comprising 1752-2382 (intron 1), 2575-3604 (intron 2) and 3770-4516 (intron 3), of SEQ ID NO:2. Features of the upstream regulatory region of the genomic DNA include a TATA box centred on bp 1487; a cap signal 32 bp down stream centred on bp 1520. Also noted within the genomic sequence are three polyadenylation signals centred on bp 4520, 4598, 4663 and a polyadenylation site at bp 4700.

This promoter is considered seed coat specific since the peroxidase protein encoded by the Ep gene accumulates in the seed coat tissues, especially in the hourglass cells of the subepidermis, and is not expressed in other tissues, aside from a marginal expression of peroxidase in the root tissues. This is also true at the transcriptional level (see FIG. 9). The DNA regulatory regions of the genomic sequence of FIG. 2 are used to control the expression of the adjacent peroxidase gene in seed coat tissue. Such regulatory regions include nucleotides 1-1532. Other regions of interest include nucleotides 1752-2382, 2575-3604 and/or 37704032 of SEQ ID NO:2. Therefore other proteins of interest may be expressed in seed coat tissues by placing a gene capable of expressing the protein of interest under the control of the DNA regulatory elements of this invention. Genes of interest include but are not restricted to herbicide resistant genes, genes encoding viral coat proteins, or genes encoding proteins conferring biological control of pest or pathogens such as an insecticidal protein for example B. thuringiensis toxin. Other genes include those capable of the production of proteins that alter the taste of the seed and/or that affect the nutritive value of the soybean.

A modified DNA regulatory sequence may be obtained by introducing changes into the natural sequence. Such modifications can be done through techniques known to one of skill in the art such as site-directed mutagenesis, reducing the length of the regulatory region using endonucleases or exonucleases, increasing the length through the insertion of linkers or other sequences of interest. Reducing the size of DNA regulatory region may be achieved by removing 3′ or 5′ regions of the regulatory region of the natural sequence by using a endonuclease such as BAL 31 (Sambrook et al 1989). However, any such DNA regulatory region must still function as a seed coat specific DNA regulatory region.

It may be readily determined if such modified DNA regulatory elements are capable of acting in a seed coat specific manner transforming plant cells with such regulatory elements controlling the expression of a suitable marker gene, culturing these plants and determining the expression of the marker gene within the seed coat as outlined above. One may also analyze the efficacy of DNA regulatory elements by introducing constructs comprising a DNA regulatory element of interest operably linked with an appropriate marker into seed coat tissues by using particle bombardment directed to seed coat tissue and determining the degree of expression of the regulatory region as is known to one of skill in the art.

Two tandemly arranged genes encoding anionic peroxidase expressed in stems of Populus kitakamiensis, prxA3a and prxA4a have been cloned and characterized (Osakabe et al, 1995). Both of these genomic sequences contained four exons and three introns and encoded proteins of 347 and 343 amino acids, respectively. The two genes encode distinct isozymes with deduced M_(r)s of 33.9 and 34.6 kDa. Furthermore, a 532 bp promoter derived from the peroxidase gene of Armoracia rusticana has also been reported (Toyobo KK, JP 4,126,088, Apr. 27, 1992). However, a search using GenBank revealed no substantial similarity between the promoter region, or introns 1, 2 and 3 of this invention and those within the literature.

Digestion of the genomic DNA with BamHI or SacI revealed restriction fragment length polymorphisms that distinguished EpEp and epep genotypes. Although the XbaI digestion did not produce a readily detectable polymorphism, the size of the hybridizing fragment in both genotypes was −14 kb. Thus, a 0.3 kb size difference is outside of the resolving power of the separation for fragments this large. Sequence analysis of EpEp and epep genotypes indicates that the mutant ep allele is missing 87 bp of sequence at the 5′ end of the structural gene. This would account for the drastically reduced amounts of peroxidase enzyme present in seed coats of epep plants since the deletion includes the translation start codon and the entire N-terminal signal sequence. However, the 87 bp deletion cannot account for the differences observed in the RFLP analysis since the missing fragment does not include a BamHI site and is much smaller than the 0.3 kb polymorphism detected in the SacI digestion. Thus, other genetic rearrangements must occur in the vicinity of the ep locus that lead to these polymorphisms.

The results shown here indicate that the mutation causing low seed coat peroxidase activity occurs in the structural gene encoding the enzyme. This mutation is an 87 bp deletion in the 5′ region of the gene encompassing the translation start site. Several different low peroxidase cultivars share a similar mutation in the same area, suggesting that the recessive ep alleles have a common origin or that the region is prone to spontaneous deletions or rearrangements.

Due to the industrial interest in soybean seed coat peroxidase, alternate sources for the production of this enzyme are needed. The DNA of this invention, encoding the seed coat soybean peroxidase under the control of a suitable promoter and expressed within a host of interest, can be used for the preparation of recombinant soybean seed coat peroxidase enzyme.

Soybean seed coat peroxidase has been characterized as a lignin-type peroxidase that has industrially significant properties ie: high activity and stability under acidic conditions; exhibits wide substrate specificity; equivalent catalytic properties to that of Phanerochaete chrysosporium ligin peroxidase (the currently preferred enzyme used for treatment of industrial waste waters (Wick 1995) but is at least 150-fold more stable; more stable than horseradish peroxidase which is also used in industrial effluent treatments and medical diagnostic kits (McEldoon et al., 1995). These properties are useful within industrial applications for the degradation of natural aromatic polymers including lignin and coal (McEldoon et al, 1995), and the preferred use of soybean peroxidase, over that of horseradish peroxidase, in medical diagnostic tests as an enzyme label for antigens, antibodies, oligonucleotide probes, and within staining techniques (Wick 1995). Soybean peroxidase is also used in the deinking of printed waste paper (Johnson et al., U.S. Pat. No. 5,270,770; Dec. 6, 1994) and for the biocatalytic oxidation of primary alcohols (Johnson et al., U.S. Pat. No. 5,391,488; Feb. 13, 1996). Soybean peroxidase has also been used as a replacement for chlorine in the pulp and paper industry, in order to remove chlorine, phenolic or aromatic amine containing pollutants from industrial waste waters (Wick 1995), or as formaldehyde replacement (Freiberg, 1995) for use in adhesives, abrasives, and protective coatings (e.g. varnish and resins, Wick 1995).

Furthermore, the seed coat peroxidase gene may be expressed in an organ or tissue specific manner within a plant. For example, the quality and strength of cotton fibber can be improved through the over-expression of cotton or horseradish peroxidase placed under the control of a fibre-specific promoter (Maliyakal, WO 95/08914; Apr. 6, 1995).

Similarly, seed-specific DNA regulatory regions of this invention may be used to control expression of genes of interest such as:

-   -   i) genes encoding herbicide resistance, or     -   ii) biological control of insects or pathogens (e.g. B.         thuringiensis), or     -   iii) viral coat proteins to protect against viral infections, or     -   iv) proteins of commercial interest (e.g. pharmaceutical), and     -   v) proteins that alter the nutritive value, taste, or processing         of seeds within the seed coat of plants.

While this invention is described in detail with particular reference to preferred embodiments thereof, said embodiments are offered to illustrate but not to limit the invention.

EXAMPLES

Plant Material

All soybean (Glycine max [L.] Merr) cultivars and breeding lines were from the collection at Agriculture Canada, Harrow, Ontario.

Seed Coat cDNA Library Construction and Screening

High seed coat peroxidase (EpEp) soybean cultivar Harosoy 63 plants were grown in field plots outdoors. Pods were harvested 35 days after flowering and seeds in the mid-to-late developmental stage were excised. The average fresh mass was 250 mg per seed. Seed coats were dissected and inmediately frozen in liquid nitrogen. The frozen tissue was lyophilized and total RNA extracted in 100 mM Tris-HCl pH 9.0, 20 mM EDTA, 4% (w/v) sarkosyl, 200 mM NaCl, and 16 mM DTT, and precipitated with LiCi using the standard phenol/chloroform method described by Wang and Vodkin (1994). The poly(A)+ RNA was purified on oligo(dT) cellulose columns prior to cDNA synthesis, size selection, ligation: into the λ ZAP Express vector, and packaging according to instructions (Stratagene). A degenerate oligonucleotide with the 5′ to 3′ sequence of TT(C/T)CA(C/T)GA(C/T)TG(C/T)TT(C/T)GT was 5′ end labelled to high specific activity and used as a probe to isolate peroxidase cDNA clones (Sambrook et al., 1989). Duplicate plaque lifts were made to nylon filters (Amersham), UV fixed, and prehybridized at 36° C. for 3 h in 6×SSC, 20 mM Na₂HPO₄ (pH6.8), 5× Denhardt's, 0.4% SDS, and 500 μg/mL salmon sperm DNA. Hybridization was in the same buffer, without Denhardt's, at 36° C. for 16 h. Filters were washed quickly with several changes of 6×SSC and 0.1% SDS, first at room temperature and finally at 40° C., prior to autoradiography for 16 h at −70° C. with an intensifying screen.

Genomic DNA Isolation, Library Construction, and DNA Blot Analysis

Soybean genomic DNA was isolated from leaves of greenhouse grown plants or from etiolated seedlings grown in vermiculite. Plant tissue was frozen in liquid nitrogen and lyophilized before extraction and purification of DNA according to the method of Dellaporta et al. (1983). Restriction enzyme digestion of 30 μg DNA, separation on 0.5% agarose gels and blotting to nylon membranes followed standard protocols (Sambrook et al., 1989). For construction of the genomic library, DNA purified from Harosoy 63 leaf tissue was partially digested with BamHI and ligated into the λ FIX II vector (Stratagene). Gigapack XL packaging extract (Stratagene) was used to select for inserts of 9 to 22 kb. After library amplification, duplicate plaque lifts were hybridized to cDNA probe.

Blots or filter lifts were prehybridized for 2 h at 65° C. in 6×SSC, 5× Denhardt's, 0.5% SDS, and 100 μg/mL salmon sperm DNA. Radiolabelled cDNA probe (20 to 50 ng) was prepared using the Ready-to-Go labelling kit (Pharmacia) and ³²P-dCTP (Amersham). Unincorporated P-dCTP was removed by spin column chromatography before adding radiolabelled cDNA to the hybridization buffer (identical to prehybridization buffer without Denhardt's). Hybridization was for 20 h at 65° C. Membranes were washed twice for 15 min at room temperature with 2×SSC, 0.5% SDS, followed by two 30 min washes at 65° C. with 0.1×SSC, 0.5% SDS. Autoradiography was for 20 h at −70° C. using an intensifying screen and X-OMAT film (Kodak).

DNA Sequencing

Sequencing of DNA was performed using dye-labelled terminators and Taq-FS DNA polymerase (Perkin-Elmer). The PCR protocol consisted of 25 cycles of a 30 sec melt at 96° C., 15 sec annealing at 50° C., and 4 min extension at 60° C. Samples were analyzed on an Applied Biosystems 373A Stretch automated DNA sequencer.

Polymerase Chain Reaction

PCR amplifications contained 1 ng template DNA, 5 pmol each primer, 1.5 mM MgCl₂, 0.15 mM deoxynucleotide triphosphates mix, 10 mM Tris-HCl, 50 mM KCl, pH 8.3, and 1 unit of Taq polymerase (Gibco BRL) in a total volume of 25 μL. Reactions were performed in a Perkin-Elmer 480 thermal cycler. After an initial 2 min denaturation at 94° C., there were 35 cycles of 1 min denaturation at 94° C., 1 min annealing at 52° C., and 2 min extension at 72° C. A final 7 min extension at 72° C. completed the program. The following primers were used for PCR analysis of genomic DNA: prx2+ CTTCCAAATATCAACTCAAT prx6− TAAAGTTGGAAAAGAAAGTA prx9 ATGCATGCAGGTTTTTCAGT prx10− TTGCTCGCTTTCTATTGTAT prx12+ TCTTCGATGCTTCTTTCACC prx29+ CATAAACAATACGTACGTGAT

RNA Isolation

For isolation of RNA, tissue was harvested from greenhouse grown plants, dissected, frozen in liquid nitrogen, and lyophilized prior to extraction. Total RNA was purified from seed coats, embryos, pods, leaves, and flowers using standard phenol/chloroform method (Sambrook et al., 1989). This method did not afford good yields of RNA from roots, therefore this tissue was extracted with Triazole reagent (GibcoBRL) and total RNA purified according to manufacturers' instructions with an additional phenol-chloroform extraction step. The amount of RNA was estimated by measuring absorbance at 260 and 280 nm, and by electrophoretic separation in formaldehyde gels followed by staining with ethidium bromide and comparison to known standards. Total RNA (10 μg per sample) was prepared, subject to electrophoresis through a 1% agarose gel containing formaldehyde, and then stained with ethidium bromide to ensure equal loading of samples. The gel was blotted to nylon (Hybond>N, Amersham) according to standard methods and the RNA was fixed to the membrane by UV cross linking.

Seed Coat Peroxidase Assays

The F₃ seed was measured for peroxidase activity to score the phenotype of the F₂ population because the seed testa is derived from maternal tissue. The seeds were briefly soaked in water and the seed coat was dissected from the embryo and placed in a vial. Ten drops (˜500 μL) of 0.5% guaiacol was added and the sample was left to stand for 10 min before adding one drop (˜50 μL) of 0.1% H₂O₂. An immediate change in colour of the solution, from clear to red, indicates a positive result and high seed coat peroxidase activity.

EXAMPLE 1 The Seed Coat Peroxidase cDNA and Genomic DNA Sequences

To isolate the seed coat peroxidase transcript, a cDNA library was constructed from developing seed coat tissue of the EpEp cultivar Harosoy 63. The primary library contained 106 recombinant plaque forming units and was amplified prior to screening. A degenerate 17-mer oligonucleotide corresponding to the conserved active site domain of plant peroxidases was used to probe the library. In screening 10,000 plaque forming units, 12 positive clones were identified. The cDNA insert size of the clones ranged from 0.5 to 2.5 kb, but six clones shared a common insert size of 1.3 kb. These six clones (soyprx03, soyprx05, soyprx06, soyprx11, soy prx12, and soyprx14) were chosen for further characterization since the 1.3 kb insert size matched the expected peroxidase transcript size. Sequence analysis of the six clones showed that they contained identical cDNA transcripts encoding a peroxidase and that each resulted from an independent cloning event since the junction between the cloning vector and the transcript was different in all cases.

Since it was not clear that the entire 5′ end of the cDNA transcript was complete in any of the cDNA clones isolated, the structural gene corresponding to the seed coat peroxidase was isolated from a Harosoy 63 genomic library. A partial BamHI digest of genomic DNA was used to construct the library and more than 106 plaque forming units were screened using the cDNA probe. A positive clone, G25-2-1-1-1, containing a 17 kb insert was identified and a 4.7 kb region encoding the peroxidase was sequenced SEQ ID NO:2. This region includes 1532 nucleotides of the 5 region of the peroxidase gene.

The genomic sequence matched the cDNA sequence except for three introns encoded within the gene. The genomic sequence also revealed two additional translation start codons, beginning one bp and 10 bp upstream from the 5′ end of the longest cDNA transcript isolated. FIG. 1 shows the deduced cDNA sequence. The open reading frame of 1056 bp encodes a 352 amino acid protein of 38,106 Da. A heme-binding domain, a peroxidase active site signature sequence, and seven potential N-glycosylation sites were identified from the deduced amino acid sequence. The first 26 amino acid residues conform to a membrane spanning domain. Cleavage of this putative signal sequence releases a mature protein of 326 residues with a mass of 35,377 Da and an estimated pI of 4.4.

Relevant features of the genomic fragment (FIG. 2) include four exons at bp 192-411 (exon 1; 1533-1751 of SEQ ID NO:2), 1042-1233 (exon 2; 2383-2574 of SEQ ID NO:2), 2263-2429 (exon 3; 4033-4516 fo SEQ ID NO:2) and 2692-3174 (exon 4; 1752-2382 of SEQ ID NO:2) and three introns at bp 412-1041 (intron 1; 1752-2382 of SEQ ID NO:2), 1234-2263 (intron 2; 2575-3604 of SEQ ID NO:2) and 2430-2691 (intron 3; 3770-4032 of SEQ ID NO:2). The 1532 bp regulatory region of the genomic DNA include a TATA box centred on bp 1487 and a cap signal 32 bp down stream centred at bp 1520 of SEQ ID NO:2. Also noted within the genomic sequence are three polyadenylation signals centred on bp 4520, 4598, 4700 and a polyadenylation site at bp 4700 of SEQ ID NO:2.

FIG. 3 illustrates the relationship between the soybean seed coat peroxidase and other selected plant peroxidases. The soybean sequence is most closely related to four peroxidase cDNAs isolated from alfalfa, (see FIG. 3) sharing from 65 to 67% identity at the amino acid level with the alfalfa proteins (X90693, X90694, X90692, el-Turk et al 1996; L36156, Abrahans et al 1994). When compared with other plant peroxidases, soybean seed coat peroxidase exhibits from 60 to 65% identity with poplar (D30653 and D30652, Osakabe et al 1994)) and flax (Lo554, Omann and Tyson 1995); 50 to 60% identity with horseradish (M37156, Fujiyama et al. 1988), tobacco (D11396, Osakabe et al 1993), and cucumber (M91373, Rasmussen et al. 1992); and 49% identity with barley (L36093, Scott-Craig et al. 1994), wheat (X85228, Baga et al 1995) and tobacco (L02124, Diaz-De-Leon et al 1993) peroxidases.

A comparison of the promoter region, 1-1532 of SEQ ID NO:2, indicates that there are no similar sequences present within the GENBANK database.

EXAMPLE 2 DNA Blot Analysis Using the Seed Coat Peroxidase cDNA Probe Reveals Restriction Fragment Length Polymorphisms Between EpEp and epep Genotypes

Genomic DNA blots of OX347 (EpEp) and OX312 (epep) plants were hybridized with ³²P-labelled cDNA to estimate the copy number of the seed coat peroxidase gene and to determine if this locus is polymorphic between the two genotypes. FIG. 4 shows the hybridization patterns after digestion with BamHI, XbaI, and SacI. Restriction fragment length polymorphisms are clearly visible in the BamHI and SacI digestions. The BamHI digestion produced a strongly hybridizing 17 kb fragment and a faint 3.4 kb fragment in the EpEp genotype. The 3.4 kb BamHI fragment is visible in the epep genotype but the 17 kb fragment has been replaced by a signal at >20 kb. The SacI digestion resulted in detection of three fragments in EpEp and epep plants. At least two fragments were expected here since the cDNA sequence has a SacI site within the open reading frame. However, the smallest and most strongly hybridizing of these fragments is 5.2 kb in EpEp plants and 4.9 kb in epep plants.

Digestion with XbaI produced hybridizing fragments of −14 kb and 7.8 kb for both genotypes, with the larger fragment showing a stronger signal.

EXAMPLE 3 A Deletion Mutation Occurs in the Recessive ep Locus

The structural gene encoding the seed coat peroxidase is schematically illustrated in FIG. 5. The 17 kb BamHI fragment encompassing the gene includes 191 bp of sequence upstream from the translation start codon, three introns of 631 bp, 1030 bp, and 263 bp, and 13 kb of sequence downstream from the polyadenylation site. The arrangement of four exons and three introns and the placement of introns within the sequence is similar to that described for other plant peroxidases (Simon, 1992; Osakabe et al. 1995).

Primers were designed from the DNA sequence to compare EpEp and epep genotypes by PCR analysis. FIG. 6 shows PCR amplification products from four different primer combinations using OX312 (epep) and OX347 (EpEp) genomic DNA as template. The primer annealing site for prx29+begins 182 bp upstream from the ATG start codon; the remaining primer sites are shown in FIG. 1. Amplification with primers prx2+ and prx6−, and with prx12+ and prx10− produced the expected products of 1.9 kb and 860 bp, respectively, regardless of the Eplep genotype of the template DNA. However, PCR amplification with primers prx9+ and prx10−, and with prx29+ and prx10− generated the expected products only when template DNA was from plants carrying the dominant Ep allele. When template DNA was from an epep genotype, no product was detected using primers prx9+ and prx10− and a smaller product was amplified with primers prx29+ and prx10−. The products resulting from amplification of OX312 or OX347 template DNA with primers prx29+ and prx10− were directly sequenced and compared. The polymorphism is due to an 87 bp deletion occurring within this DNA fragment in OX312 plants, as shown in FIG. 5. This deletion begins nine bp upstream from the translation start codon and includes 78 bp of sequence at the 5′ end of the open reading frame, including the prx9+ primer annealing site.

To test whether this deletion mutation cosegregates with the seed coat peroxidase phenotype, genomic DNA from an F₂ population segregating at the Ep locus was amplified using primers prx9+ and prx10− and F₃ seed was tested for seed coat peroxidase activity. FIG. 7 shows the results from this analysis. Of the 30 F₂ individuals tested, all 23 that were high in seed coat peroxidase activity produced the expected 860 bp PCR amplification product. The remaining seven F₂'s with low seed coat peroxidase activity produced no detectable PCR amplification products.

Finally, to determine if the OX312(epep) and OX347(EpEp) breeding lines are representative of soybean cultivars that differ in seed coat peroxidase activity, several cultivars were tested by PCR analysis using primer combinations targeted to the Ep locus. FIG. 8 shows results from this analysis of six different soybean cultivars, three each of the homozygous dominant EpEp and recessive epep genotypes. As observed with OX312 and OX347, amplification products of the expected size were produced with primers prx12+ and prx10− regardless of the genotype, whereas epep genotypes yielded no product with primers prx9+ and prx10− or a smaller fragment with primers prx29+ and prx10−.

EXAMPLE 4 Developmental Pattern of Expression of the Ep Gene

The seed coat peroxidase mRNA levels were determined by hybridizing RNA gel blots with radio labelled cDNA probe. The figure illustrates the transcript abundance in various tissues of epep and EpEp plants. The mRNA accumulated to high levels in seed coat tissues of EpEp plants, especially in the later stages development when whole seed fresh weight exceeded 50 mg. Low levels of transcript could also be detected in root tissues but not in the flower, embryo, pod or leaf. The transcript could also be detected in seed coat and root tissues epep plants but in drastically reduced amounts compared to the EpEp genotype. The reduced amounts of peroxidase mRNA present in seed coats of epep plants indicates that the transcriptional process and/or the stability of the resulting mRNA is severely affected. The Ep gene has a TATA box and a 5′ cap signal beginning 47 bp and 15 bp, respectively, upstream from the translation start codon. The 87 bp deletion in the ep allele extends into the 5′ cap signal and therefore could interfere with transcript processing. Regardless, any resulting transcript will not be properly translated since the AUG initiation codon and the entire amino-terminal signal sequence is deleted from the ep allele. Not wishing to be bound by theory, the lack of peroxidase accumulation in seed coats of epep plants appears to be due to at least two factors, greatly reduced transcript levels and ineffective translation, resulting from mutation of the structural gene encoding the enzyme. In summary, the results indicate that the Ep gene regulatory elements can drive high level expression in a tightly coordinated, tissue and developmentally specific manner.

All scientific publications and patent documents are incorporated herein by reference.

The present invention has been described with regard to preferred embodiments. However, it will be obvious to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as described in the following claims.

REFERENCES

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1. (canceled)
 2. An isolated DNA molecule comprising at least 24 contiguous nucleotides selected from nucleotides 1-1532 of SEQ ID NO:2. 3-29. (canceled)
 30. A chimeric DNA molecule, said DNA molecule comprising a segment of a plant expressible promoter, providing a TATA box region and a transcription start site, whereby a heterologous plant expressible structural gene, placed 3′ to said segment of the plant expressible promoter is expressed under the regulatory control of said segment of the plant expressible promoter, and wherein said segment of the plant expressible promoter is selected from the group consisting of: a) a nucleotide sequence consisting of nucleotides 9 to 1532 of SEQ ID NO:2; b) a nucleotide sequence consisting of nucleotides 459 to 1532 of SEQ ID NO:2; c) a nucleotide sequence consisting of nucleotides 1066 to 1532 of SEQ ID NO:2; and d) a nucleotide sequence consisting of nucleotides 1326 to 1532 of SEQ ID NO:2. 